Phytases are myo-inositol hexakisphosphate phosphohydrolases that catalyze the stepwise removal of inorganic orthophosphate from phytate (myo-inositol hexakisphosphate) (1). There are two types of phytases. One is called 3-phytase (EC.3.1.3.8) which initiates the removal of phosphate groups at the positions 1 and 3 of the myo-inositol ring. The other is called 6-phytase (EC.3.1.3.26) which first frees the phosphate at the position 6 of the ring. While no phytase has been identified from animal tissues, plants usually contain 6-phytases and a broad range of microorganisms, including bacteria, filamentous fungi, and yeast, produce 3-phytases (2-9). Because over 70% of the total phosphorus in foods or feeds of plant origin is in the form of phytate that is poorly available to simple-stomached animals and humans, phytases are of great uses in improving mineral nutrition of these species (10-16). Supplemental microbial phytases in diets for swine and poultry effectively enhance bioavailability of phytate phosphorus and reduce the need for inorganic phosphorus supplementation (11-15), resulting less phosphorus pollution in areas of intensive animal production (8-15). However, a relatively high level of phytase supplementation is necessary in animal diets (10-16), because a considerable amount of the enzyme is degraded in stomach and small intestine (13), probably by proteolysis of pepsin and trypsin. Meanwhile, the proteolytic profiles of various phytases were not studied. Clearly, a better understanding of their sensitivities to trypsin and pepsin hydrolysis could be helpful for improving the nutritional value of phytases. Aspergillus niger phytase gene (phyA) has been overexpressed in its original host (17) and the recombinant enzyme (r-PhyA, EC 3.1.3.8) has been used in animal diets as a commercial phytase (13, 14). This enzyme is a glycoprotein of approximately 80 kDa. Escherichia coli pH 2.5 acid phosphatase gene (appA) has also been characterized (18, 19). Animal experiments have demonstrated that the recombinant enzyme (r-AppA, EC: 3.1.3.2) is as effective as r-PhyA in releasing phytate phosphorus in animal diets (14).
But, expenses of the limited available commercial phytase supply and the activity instability of the enzyme to heat of feed pelleting preclude its practical use in animal industry. Therefore, there is a need for enzymes which have a high level of phytase activity and a high level of stability for use in animal feed.
The present invention provides a phosphatase fragment having improved phytase activity. A fragment of a phosphatase having increased phytase activity is produced by treating the phosphatase with a protease.
The invention further provides a recombinant gene encoding a phosphatase fragment having improved phytase activity. The vector consists of a promoter, a coding region encoding the phosphatase fragment, and a terminator.
In another embodiment, the invention provides a method of increasing the phytase activity of phosphatase by treating the phosphatase with a protease.
The invention also provides a phosphatase having improved phytase activity, which has an amino acid sequence as shown in SEQ. ID No. 1.